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A duplex RT-PCR primer, kit and method for simultaneous amplification of North American and European porcine PRRS virus

A porcine blue ear disease virus, RT-PCR technology, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of indistinguishability, infection, etc., and achieve strong specificity and high sensitivity Effect

Active Publication Date: 2020-01-03
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the same pig in the same farm has already been infected with both European PRRS and North American PRRS. It is basically indistinguishable clinically, which brings great challenges to prevention and treatment.

Method used

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  • A duplex RT-PCR primer, kit and method for simultaneous amplification of North American and European porcine PRRS virus
  • A duplex RT-PCR primer, kit and method for simultaneous amplification of North American and European porcine PRRS virus
  • A duplex RT-PCR primer, kit and method for simultaneous amplification of North American and European porcine PRRS virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Primer Design

[0045] According to the genome sequences of North American and European pig PRRS virus in GenBank, 2 pairs of primers were designed respectively, among which NA-ORF5-F and NA-ORF5-R primers can cover more North American pig PRRS to the greatest extent virus (including North American PRRS classic strains, variant strains and vaccine strains); EU-ORF5-F and EU-ORF5-R primer pairs can cover more European PRRS viruses (including vaccine strains and field strains). Its base sequence is shown below.

[0046] NA-ORF5-F: 5 ， -GTTTTAGCCTGTCTTTTTTGC-3 ， (SEQ ID NO: 1);

[0047] NA-ORF5-R:5 ， -GCTGAGTACACHCCCCAAAG-3 ， (SEQ ID NO: 2);

[0048] EU-ORF5-F: 5 ， -ACCATYGCTTGTTTGTTCGCCAT-3 ， (SEQ ID NO: 3);

[0049] EU-ORF5-R: 5 ， -CTTTTCCACAGCGTATGCTCG-3 ， (SEQ ID NO: 4).

Embodiment 2

[0050] Embodiment 2 Double RT-PCR detection method

[0051] 1) Extraction of RNA

[0052] Viral nucleic acid was extracted according to the instructions of Axygen's DNA / RNA extraction kit.

[0053] 2) Double RT-PCR amplification reaction

[0054] The double RT-PCR method was established by using the positive porcine PRRS virus nucleic acid isolated clinically and identified as mixed infection of North American type and European type as the template of double RT-PCR. Use 50 μL RT-PCR reaction system: PrimeScript 1 stepEnzyme Mix 1 μL, 2×1 step Buffer 25 μL, primers NA-ORF5-F, NA-ORF5-R, EU-ORF5-F and EU-ORF5-R 1 μL each , viral nucleic acid template 6 μL, RNase Free ddH 2 O 14 µL for a total volume of 50 µL.

[0055] RT-PCR reaction program: reverse transcription at 50°C for 30 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 60 s, a total of 35 cycles; final extension at 72°C for 5 min.

[0056] Obse...

Embodiment 3

[0057] Embodiment 3 specificity test

[0058] According to the method of embodiment 2, utilize double RT-PCR method to the PRRSV and porcine circovirus type 2 virus, swine fever virus, porcine foot-and-mouth disease virus, porcine pseudorabies virus, porcine parvovirus, porcine parvovirus, Porcine Kubu virus and porcine D-coronavirus were tested.

[0059] see results figure 1 . Lane M: DNA Marker 2000; Lane 1: North American + European PRRSV; Lane 2: European PRRSV; Lane 3: North American PRRSV; Lane 4: Porcine circovirus type 2; Lane 5: Classical swine fever virus; Lane 6 : porcine foot-and-mouth disease virus; lane 7: porcine pseudorabies virus; lane 8: porcine parvovirus; lane 9: porcine Kubu virus;

[0060] Depend on figure 1 It can be seen that the optimized double RT-PCR detection method was used to amplify the positive nucleic acid of PRRSV mixed infection of North American type and European type, and the results obtained fragments of about 900bp and 1036bp in size,...

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Abstract

The invention discloses double RT-PCR (reverse transcription-polymerase chain reaction) primer, kit and method for amplifying North America and European porcine blue ear disease viruses; the method has the advantages of high specificity, high sensitivity, low time and labor consumption and the like, and is of important guidance significance to molecular epidemiology, genetic evolution, vaccine research and development and the like for studying porcine blue ear disease. In addition, denaturing and annealing temperature in the method only takes 20 s, amplification can be performed as well just with one PCR apparatus, and the method may also act as a method for identifying and detecting North America and European porcine blue ear disease viruses and helps quickly classify porcine blue ear viruses.

Description

technical field [0001] The invention belongs to the detection field, and in particular relates to a double RT-PCR primer, a kit and a method for simultaneously amplifying North American and European porcine PRRS viruses. Background technique [0002] Pig blue ear disease is also known as porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS), and its pathogen is porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV). Since the disease was discovered in the United States in 1987, the disease has spread rapidly to the pig industry all over the world, bringing huge economic losses to the pig industry. So far, PRRS has not only brought a catastrophic impact on my country's pig industry, but also broke out in neighboring countries such as Vietnam and South Korea. It can be seen that the prevalence of PRRS is increasing. Pigs of various breeds and ages can be infected with PRRS....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 罗满林周霞翟少伦张贺魏文康吕殿红温肖会刘建奎李冰翟俊琼邹舒展吴梦矾
Owner SOUTH CHINA AGRI UNIV
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